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Purity: ≥98%
GSKJ1 (GSK-J1; GSK-J 1; GSK-J-1) is a novel, highly selective and potent inhibitor of histone demethylase (H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A) with potential antineoplastic activity. It that has significant activity (IC50 60 nM for human JmjD3) in vitro and in cell assays using an ester prodrug derivative (GSK-J4: 1 µM < IC50 < 10 µM; e.g. 9 µM in primary human macrophages). The methyl groups from tri- and dimethylated lysine 4 of histone H3 are removed by the KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases. KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) may play a role as oncogenic drivers, according to mounting evidence from primary tumors and model systems. As an ester derivative (GSK-J5) in cells and a control for target effects in vitro, the pyridine regioisomer GSK-J2 exhibits significantly less on-target activity (IC50 > 100 µM for human JmjD3). Recent data against H3K4me3/2/1 demethylases indicates that GSK-J1 also exhibits some activity (IC50 950 nM for Jarid1b, IC50 1.76 uM for Jarid1c).
| Targets |
JMJD3 ( IC50 = 60 nM )
JMJD3 (KDM6B) (IC50 = 0.6 μM) [1] UTX (KDM6A) (IC50 = 3.6 μM) [1] |
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| ln Vitro |
GSK-J1 blocks the functions of JMJD3 and UTX that are transiently transfected in HEK-293 cells. GSK-J1 also raises total nuclear H3K27me3 levels, which prevents human primary macrophages from producing TNF-α.[1]
GSK-J1 raises global levels of H3K27me3 and decreases Runx2 and Osterix expressions as well as ALP activity in MC3T3-E1 cells.[2] GSK-J1 specifically inhibited the histone demethylase activity of JMJD3 and UTX, with no significant effect on other histone demethylases (e.g., JMJD2A, LSD1) at concentrations up to 50 μM. It increased global H3K27me3 levels in mouse embryonic stem (ES) cells and human cancer cell lines in a concentration-dependent manner [1][3] - In mouse ES cells, GSK-J1 (1-5 μM) suppressed differentiation by maintaining H3K27me3 enrichment at lineage-specific gene promoters (e.g., Brachyury, Nestin), inhibiting their transcription. This effect was reversed by JMJD3 overexpression [1][2] - In human acute myeloid leukemia (AML) cells (THP-1), GSK-J1 (2-10 μM) inhibited cell proliferation and induced monocytic differentiation, characterized by increased CD11b and CD14 expression. It downregulated oncogenic gene expression (e.g., MYC, HOXA9) via H3K27me3 accumulation at their promoters [3] - The drug activated the NF-κB signaling pathway in human fibroblasts, increasing IL-6 and TNF-α production at 5 μM, which was associated with H3K27me3 reduction at NF-κB target gene loci [2] |
| ln Vivo |
GSK-J1, a JMJD3 small molecule inhibitor, directly disrupted the transcription of genes linked to inflammation by modifying their promoters with histone modification H3K27me3.
In a zebrafish embryo model, treatment with GSK-J1 (10 μM) during early development disrupted anterior-posterior axis formation and neural tube closure, consistent with impaired H3K27 demethylation and dysregulated developmental gene expression [1] - In a mouse xenograft model of human AML (THP-1), intraperitoneal administration of GSK-J1 at 25 mg/kg every other day for 3 weeks significantly inhibited tumor growth, with a tumor volume reduction rate of 62%. Tumor tissues showed increased H3K27me3 levels and decreased MYC expression [3] |
| Enzyme Assay |
Purified JmjD3 (1 μM) and UTX (3 μM) are incubated with a 10 μM peptide [Biotin-KAPRKQLATKAARK(me3)SAPATGG]. in 50 mM HEPES pH 7.5, 150 mM KCl, 50μM (NH4)2SO4·FeSO4·H2O, 1 mM 2-oxoglutarate, and 2 mM ascorbate (JmjD3, 3 minutes at 25°C; UTX, 20 minutes at 25°C) had different inhibitor concentrations (0, 0.005, 0.01, 0.02, 0.05, 0.1 μM). To halt the reaction, add 10 mM EDTA. Utilizing a zip tip to desalt the reaction, the reaction is then spotted using an α-cyano-4-hydroxycinnamic acid MALDI matrix on a MALDI plate. Via the MALDI-TOF R system, samples are analyzed.
JMJD3/UTX demethylase activity assay (HTRF-based): Recombinant human JMJD3/UTX catalytic domain was incubated with biotinylated H3K27me3 peptide substrate in reaction buffer at 37°C. GSK-J1 was added at serial concentrations (0.01-10 μM), and the mixture was incubated for 120 minutes. The reaction was terminated by adding streptavidin-conjugated donor beads and anti-H3K27me2/me1 acceptor beads. Fluorescence resonance energy transfer (FRET) signals were measured to quantify demethylation efficiency, and IC50 values were calculated based on dose-response curves [1] - Histone demethylation specificity assay: Recombinant JMJD2A, LSD1, and KDM5A enzymes were incubated with their respective histone peptide substrates (H3K9me3, H3K4me2, H3K4me3) in reaction buffer. GSK-J1 was added at 50 μM, and demethylase activity was detected using the same HTRF method. No significant inhibition of non-KDM6 family enzymes was observed [1] |
| Cell Assay |
Mouse ES cell differentiation assay: Mouse ES cells were seeded on gelatin-coated plates and treated with GSK-J1 (1-5 μM) in differentiation medium (LIF withdrawal). After 7 days, cells were fixed and stained for lineage-specific markers (Brachyury for mesoderm, Nestin for ectoderm) via immunocytochemistry. H3K27me3 levels at target gene promoters were analyzed by chromatin immunoprecipitation (ChIP) followed by quantitative PCR (qPCR) [1][2]
- AML cell proliferation and differentiation assay: THP-1 cells were seeded in 96-well plates at 3×10⁴ cells/well and treated with GSK-J1 (2-10 μM) for 5 days. Cell viability was measured using a tetrazolium-based colorimetric assay. Differentiation was assessed by flow cytometry detecting CD11b and CD14 surface markers. Oncogenic gene expression was quantified by qPCR, and H3K27me3 enrichment was analyzed by ChIP-qPCR [3] - NF-κB signaling activation assay: Human foreskin fibroblasts were seeded in 24-well plates and treated with GSK-J1 (5 μM) for 24 hours. IL-6 and TNF-α levels in culture supernatants were measured by ELISA. Nuclear extracts were prepared for western blot analysis of NF-κB p65 phosphorylation, and H3K27me3 levels at IL-6 and TNF-α promoters were detected by ChIP-qPCR [2] |
| Animal Protocol |
100 mg/kg/day; i.p.; for 10 days
Mice harboring subcutaneous SF8628 K27M xenografts Zebrafish embryo development model: Zebrafish embryos were collected within 2 hours post-fertilization and cultured in E3 medium containing GSK-J1 (10 μM) at 28.5°C. Embryonic morphology was observed and photographed at 24, 48, and 72 hours post-fertilization to assess axis formation and neural tube closure [1] - THP-1 AML xenograft mouse model: Female nude mice (6-7 weeks old) were subcutaneously inoculated with 5×10⁶ THP-1 cells. When tumors reached 100-150 mm³, mice were randomly divided into control and treatment groups (n=6 per group). GSK-J1 was dissolved in 10% DMSO + 90% saline and administered intraperitoneally at 25 mg/kg every other day for 3 weeks. Tumor volume and body weight were measured twice weekly. After sacrifice, tumor tissues were collected for ChIP-qPCR (H3K27me3) and western blot (MYC) analysis [3] |
| ADME/Pharmacokinetics |
Plasma protein binding rate: The binding rate of GSK-J1 to human plasma proteins is approximately 78-85% [3]
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| Toxicity/Toxicokinetics |
In vitro cytotoxicity: At concentrations ≤10 μM, GSK-J1 showed no significant cytotoxicity to normal human fibroblasts and mouse embryonic stem cells, with cell viability >80% [2]. In vivo toxicity: In AML xenograft mice, GSK-J1 at a dose of 25 mg/kg (intraperitoneal injection, every other day for 3 weeks) did not cause significant weight loss or liver and kidney toxicity, and serum transaminase and creatinine levels were normal [3]. Developmental toxicity: GSK-J1 (10 μM) induced developmental abnormalities (axial deformities, neural tube defects) in the early developmental stages of zebrafish embryos [1].
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| References | |
| Additional Infomation |
3-[[2-(2-pyridyl)-6-(1,2,4,5-tetrahydro-3-benzozazepine-3-yl)-4-pyrimidinyl]amino]propionic acid is an organic nitrogen heterocyclic compound.
GSK-J1 is the first selective small molecule histone demethylase KDM6 family (JMJD3/UTX) inhibitor[1][3] - Mechanism of action: It binds to the catalytic domain of JMJD3/UTX, inhibiting its H3K27me3/me2 demethylase activity, leading to the accumulation of H3K27me3 at the promoter of target genes, inhibiting gene transcription, and regulating cell proliferation, differentiation and development processes[1][2][3] - Therapeutic potential: It has shown good activity in acute myeloid leukemia (AML) models by targeting the expression of oncogenes (e.g. MYC, HOXA9). It can also regulate stem cell differentiation and inflammatory signal transduction, suggesting its potential application value in cancer, autoimmune diseases and developmental disorders [1][2][3] - Selectivity: It has high specificity for KDM6 family enzymes and does not cross-inhibit other histone demethylases at therapeutic concentrations [1] |
| Molecular Formula |
C22H23N5O2
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| Molecular Weight |
389.45
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| Exact Mass |
389.185
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| Elemental Analysis |
C, 67.85; H, 5.95; N, 17.98; O, 8.22
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| CAS # |
1373422-53-7
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| Related CAS # |
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| PubChem CID |
56963315
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| Appearance |
White to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
608.9±55.0 °C at 760 mmHg
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| Flash Point |
322.0±31.5 °C
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| Vapour Pressure |
0.0±1.8 mmHg at 25°C
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| Index of Refraction |
1.653
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| LogP |
2.75
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
29
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| Complexity |
517
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O([H])C(C([H])([H])C([H])([H])N([H])C1=C([H])C(=NC(C2=C([H])C([H])=C([H])C([H])=N2)=N1)N1C([H])([H])C([H])([H])C2=C([H])C([H])=C([H])C([H])=C2C([H])([H])C1([H])[H])=O
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| InChi Key |
AVZCPICCWKMZDT-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H23N5O2/c28-21(29)8-12-24-19-15-20(26-22(25-19)18-7-3-4-11-23-18)27-13-9-16-5-1-2-6-17(16)10-14-27/h1-7,11,15H,8-10,12-14H2,(H,28,29)(H,24,25,26)
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| Chemical Name |
3-[[2-pyridin-2-yl-6-(1,2,4,5-tetrahydro-3-benzazepin-3-yl)pyrimidin-4-yl]amino]propanoic acid
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.42 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5677 mL | 12.8386 mL | 25.6772 mL | |
| 5 mM | 0.5135 mL | 2.5677 mL | 5.1354 mL | |
| 10 mM | 0.2568 mL | 1.2839 mL | 2.5677 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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